Environ. Sci. Technol. 2008, 42, 1967–1972
Biotransformation Enzymes and Thyroid Axis Disruption in Juvenile Rainbow Trout (Oncorhynchus mykiss) Exposed to Hexabromocyclododecane Diastereoisomers V I N C E P . P A L A C E , * ,†,‡ K E R R I P L E S K A C H , † THOR HALLDORSON,† ROBERT DANELL,† KERRY WAUTIER,† BOB EVANS,† MEHRAN ALAEE,§ CHRIS MARVIN,§ AND G R E G G T . T O M Y †,| Department of Fisheries & Ocean, Freshwater Institute, Winnipeg, Manitoba, R3T 2N6 Canada, Department of Zoology and Departments of Chemistry, Environmental Science, and Geography, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 Canada, and Environment Canada, National Water Research Institute, Burlington, Ontario, L7R 4A6 Canada
Received October 10, 2007. Revised manuscript received December 13, 2007. Accepted December 17, 2007.
Juvenile rainbow trout (Oncorhynchus mykiss) were fed either a reference diet or one of three diets enriched with R, β, or γ diastereoisomers of hexabromocyclododecane (HBCD, C12H18Br6) for 56 days. This exposure period was followed by 112 days during which all fish were fed the reference diet. Potential effects of HBCD on phase I and II biotransformation enzyme activities and thyroid axis disruption were examined. Disruption of the thyroid axis was most evident in the γ-HBCD exposed group, as indicated by lower circular FT4 and higher FT3 as well as an increase in thyroid epithelial cell height. However, fish fed the R-HBCD enriched diet also exhibited altered glucuronyltransferase activity and thyroid epithelial cell heights and the β-HBCD group had altered FT4 and FT3 and glucuronyltransferase activity. T4ORD activity was not affected after 14 days, but was significantly lower among all HBCD exposed fish compared to the reference fish after 56 days. Results from these experiments indicate that all isomers have the potential to disrupt thyroid homeostasis.
Introduction Brominated flame retardant (BFRs) use has expanded widely over the past 20 years, with global demand for the compounds exceeding 200,000 MT in 2001 (1). Within the general BFR category, there are three more specific categories that are most common; tetrabromobisphenol-A (TBBPA), polybrominated diphenyl ethers (PBDEs), and hexabromocyclododecane (HBCD). There are important differences in global use * Author of correspondence: ph: (204)-983-5004; fax: (204)-9846587; email:
[email protected]. † Freshwater Institute. ‡ Department of Zoology, University of Manitoba. § National Water Research Institute. | Departments of Chemistry, Environmental Science, and Geography, University of Manitoba. 10.1021/es702565h CCC: $40.75
Published on Web 02/07/2008
2008 American Chemical Society
patterns of these three categories. For example, European use of TBBPA and HBCD exceed PBDEs, partly as a result of a voluntary ban of the lower brominated forms of PBDEs that was formalized in 2003 (2). In North America, PBDE demands exceed TBBPA and HBCD, but growing concern regarding potential biological effects of some PBDEs will likely move more consumer products to include alternative compounds in the near future. The primary biological concerns regarding PBDEs are their propensity to accumulate in biota (3) and their structural similarity to thyroid hormones (Figure 1.) that allows them to disrupt thyroid hormone metabolism, transport and binding kinetics (4–6). Hexabromocyclododecane is used as an additive flame retardant in polystyrene foams that are used as thermal insulators in the building industry as well in upholstery. HBCD use has increased in Europe during the past decade (3), and it is likely to in North America, as the compound replaces PBDEs. Because of its structural dissimilarity to PBDEs and thyroid hormones (Figure 1) HBCD might be expected to have lower potential to disrupt biotransformation enzymes and the thyroid axis. However, recent studies suggest that like some PBDEs, HBCD is highly bioaccumative (1, 7) and may affect biotransformation enzyme systems and thyroid homeostasis in exposed organisms (8–12). The technical HBCD formulation consists of three diastereoisomers, R, β, and γ that behave quite differently in the environment. Specifically, although the γ-form predominates in commercial formulations (>70%), the R-isomer is usually highest in the biotic environment, whereas the γ-isomer is the dominant isomer in sediment (13–15). There are currently few studies that have examined the potential for HBCD diastereisomers to affect biochemical or physiological systems (2, 12). Recent experimental evidence has shown that R- and γ- more than β-HBCD diastereoisomers are T3 agonists (12). Given the propensity for HBCD to bioaccumulate, its increasing environmental concentrations and global use patterns, and the potential for biological effects, additional data are required to assess the risks associated with the use of this compound. To address this deficiency, we held juvenile rainbow trout (Oncorhynchus mykiss) in the laboratory and fed them diets containing environmentally relevant concentrations of the individual R-, β-, and γ-isomers. Although the β-isomer is usually undetectable in the environment, it was included at a dose similar to the R- and γ-isomers for exploratory purposes. We recently reported bioaccumulation and depuration rates as well as biologically mediated conversion of R-, β-, and γ-diastereoisomers in vivo from these experiments (1). Here, we describe studies that extend on our previous work by examining the hypothesis that environmentally relevant concentrations of HBCD diastereoisomers will affect biotransformation enzymes (cytochrome P4501A (CYP1A) and glucuronyltransferase (UDPGT)) and the regulation of thyroid status (circulating free T3 and T4, thyroid gland structure and T4 outer ring deiodinase enzyme, T4ORD, activity) in rainbow trout.
Materials and Methods Summaries of the accumulation, depuration, and bioisomerization of HBCD as well as liver size data and the chemicals used in these experiments are available as Supporting Information. Food Preparation. The preparation of the fish food has been described previously (1). Briefly, four formulations of food were prepared. Three of these were spiked with a known amount of a particular diastereoisomer, whereas none of the VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Chemical structures of the thyroid hormones thyroxine (T4), triiodothyronine (T3), and general polybrominated diphenylether (PBDE) and hexabromocyclododecane (HBCD) structures. diastereoisomers were added to the reference diet. Lipidcorrected concentrations of R-, β-, γ-isomer in the food were determined to be (mean ( SEM) 29.14 ( 1.95, 11.84 ( 4.62, and 22.84 ( 2.26 ng/g, respectively. Respective lipid-corrected concentrations of R- and γ-HBCD in the control food were 0.47 and 0.84 ng/g; the β-isomer was below method detection limits. Food was stored in the dark at -4 °C to exclude the possibility of phototransformations. Fish. Animal-use protocols were approved by the Freshwater Institute Animal Care Committee, in accordance with national guidelines. Juvenile rainbow trout (Rainbow Springs Hatchery, Thamesford, ON) with initial mean weights of 233 ( 89 g were separated into four 800 L fiberglass tanks (n ) 50 in each tank) receiving water at a constant water flow of 1.5 L/min of UV- and carbon-dechlorinated Winnipeg City tap water, at a temperature of 11–12 °C and pH between 7.9 and 9.1. The dissolved oxygen was always >95% saturation. A 12 h of light, 12 h of dark photoperiod was maintained throughout the experiment. Fish were acclimatized for 7 days prior to the start of the experiment. Fish in each of the three tanks were exposed to an individual HBCD isomer via their food for an uptake phase of 56 days, followed by a depuration phase of 112 days, where all fish were fed unfortified food. The feeding rate was 1% of their body weight three times a week. This rate was adjusted after each sampling day on the basis of the mean weight of the fish sacrificed. Four fish from each tank were sacrificed on days 0, 7, 14, and 56 of the uptake phase and days 7, 14, 56, and 112 of the depuration phase. Fish were sacrificed 48 h after the previous feeding by an overdose of a pH buffered solution of tricaine methanesulfonate (0.4 g/L). Once operculum movement ceased, 3–5 mL of blood was removed via the caudal vein, along with the liver, kidney, muscle tissue, and thyroid. Blood was held on ice (7µM. Although there are mixed reports regarding the ability of HBCD to affect phase I biotransformation activity, it appears more likely that phase II, or conjugation reactions, are more relevant. Glucuronylstransferase activity was elevated in liver of fish fed the R-and β-diets, but not in the γ-group, at day 56 of the uptake phase relative to the fish fed the reference diet (Figure 4). Although no differences were evident at day 7 of the clearance phase, by day 14 fish fed the β- and γ-diet had significantly lower activity than the reference fish. This difference persisted in the γ-group to day 112 of the clearance phase. Hepatic conjugation to glucuronic acid or sulfates constitute a major excretory pathway for T4 (31). Germer et al. (11) reported that HBCD induced hepatic CYP2B and CYP3A enzymes in rats exposed to the compound via the diet, and that this effect was more pronounced in females than in males. Induction is mediated by the pregnane-X receptor (PXR) and/or the androstane receptor (CAR) in rats and phase II enzymes, including UGT, the major T4-conjugating enzyme, are also likely induced by HBCD via these receptors. If elevated conjugation was responsible for depleting circulating T4 concentrations in fish from this study, why would a concurrent decline in FT3 also not have occurred in the HBCD exposed fish? The reason may lie in the differences of T4 and T3 metabolism by conjugation enzyme systems. In rats, T3 appears to be a UGT2B3 substrate, whereas T4 is conjugated by UGT2B1 without concurrent activity toward T3 (32). This suggests that there are conjugation systems that specifically metabolize and excrete T4 without affecting T3. Jimnitz et al. (33) showed that methylcholanthrene and clofibrilate, two model UGT inducers, additively enhanced the clearance rates of T4, suggesting induction of different enzyme activities that both act on T4. They further suggested that clearance by enzymes other than UGT1A1 and UGT1A6 was also likely. Although the specific glucuronidation enzymes responsible for increasing T4 clearance from the plasma of fish exposed to HBCD remain 1970
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FIGURE 5. T4 outer ring deiodinase enzyme activity in liver of rainbow trout fed a reference diet or diets enriched in r, β, or γ individual HBCD diastereoisomers. Data are presented as mean ( SEM (n ) 4). to be fully elucidated, the mechanism probably accounts for a significant portion of the thyroid disruption in HBCD exposed organisms. In addition to glucuronidation, removal of iodines (deiodination) from thyroid hormones constitutes another major metabolic transformation to remove activity from the whole organism. Deiodinase enzyme activity is a particularly important regulator of thyroid activity in fish (26). Whereas in mammals, the thyroid gland secretes both the prohormone T4 as well as the active T3, in fish, virtually all of the T4 produced by the thyroid is converted to T3 in the peripheral tissues, primarily liver (22). There are three types of deiodinase activity in fish that correspond roughly to the D1, D2 and D3 activities described in mammalian models of the thyroid. Outer ring deiodination (T4ORD), corresponding to mammalian D2, converts T4 to the active T3 form by removing an iodine from the outer ring of the T4 molecule. The outer ring of T4 is the portion furthest from the amino acid moiety. T4ORD was assessed early in the present experiment, at day 14 of the uptake phase, and also when maximal HBCD tissue concentrations were achieved, day 56 of the uptake phase (1). This latter time coincides with the period when FT3, FT4 and thyroid glandular structure were altered. T4ORD activity was similar among all groups after 14 days of uptake, but was significantly lower among all HBCD exposed fish compared to the reference fish after 56 days of the uptake phase of the experiment (Figure 5). Higher concentrations of circulating FT3 in the HBCD exposed fish may have contributed to the lower T4ORD enzyme activity in fish from this experiment. High T3 concentrations negatively feedback to lower T4ORD by decreasing the amount of enzyme protein in liver cells (34). Hamers et al. (12) recently showed that Rand γ-HBCD, more than the β-diastereoisomer, were T3 agonists in an in vitro system. Therefore, elevated in vivo concentrations of HBCD could have contributed to the downregulation of T4ORD in this experiment. Technically, the assay used for this experiment quantifies only the 125I that is liberated from labeled T4, used as a substrate for the enzyme in the procedure. As a result, we cannot discount that deiodinase activities similar to D1 in mammals (catalalyses both outer and inner ring deiodination) and D3 (inner ring deiodination only) might have also contributed to the lower 125I that we have reported as T4ORD. However, in previous experiments where hyperthyroidism has been induced by feeding fish diets enriched with T3, it has been an activity similar to D2 that specifically converts T4 via outer ring deiodination to T3 (35), that has been most affected (31, 35–37). Furthermore, the contribution of hepatic D1 type activity to 125I-T4 deiodination is relatively small compared to D2 type hepatic activity (38) and it has been
Supporting Information Available Summaries of the accumulation, depuration, and bioisomerization of HBCD, as well as liver size data and the chemicals used in these experiments (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited
FIGURE 6. Thyroid epithelial cell heights of rainbow trout fed a reference diet or diets enriched in r, β, or γ individual HBCD diastereoisomers. Data are presented as mean ( SEM (n ) 4). suggested that it is reasonable to assume that 125I generated by this assay is representative of T4ORD in fish (26). Nevertheless, elevated T3 can further increase rates of hepatic inner ring deiodination and deiodination of T3 to T2 (26) and these considerations should be included in further work examining the effects of HBCD on deiodination rates in fish. It is unlikely that lower circulating FT4 among fish exposed to HBCD in this experiment contributed to the lower T4ORD activity. Supplementation of T4 had no effect on hepatic type II deiodinase in tilapia (Oreochromis niloticus), suggesting that it is primarily T3 that exerts control over conversion enzyme activity (35). Moreover, in hypothyroid fish, the decline in plasma T4 is most often accompanied by an increase in hepatic deiodination (39), and not a decrease, as we observed. Thyroid Histopathology. Lower circulating FT4 concentrations would be expected to stimulate TSH secretion by the pituitary by a feedback mechanism (26). This phenomenon could account for the simultaneous elevation of plasma FT3 and thyroid gland hypertrophy in fish from this study. Thyroid epithelial cell heights were significantly greater in the γ-HBCD exposed group at day 56 of the uptake phase and in fish from the R- and γ-HBCD exposed groups at day 14 of the depuration phase, relative to fish in the reference group (Figure 6). Enlarged thyroid epithelial cells are a reliable measure of thyroid gland hypertrophy (22) and in rats are one of the most sensitive effects of HBCD exposure (10). In summary, environmentally relevant concentrations of R- and γ-isomers of HBCD, and similar concentrations of β-HBCD, in the diet altered the thyroid status of juvenile rainbow trout. Lower circulating concentrations of FT4 and higher FT3 as well as altered hepatic metabolic enzymes and thyroid gland hypertrophy were evident. The data are consistent with previous studies from the mammalian literature in which a model of enhanced T4 metabolism results in positive feedback to activate the thyroid gland. Disruption of the thyroid axis was transient and alterations to circulating hormone concentrations, conjugation enzymes, and thyroid glandular structure were most evident when HBCD concentrations were highest in fish from this experiment. Glucuronidation and deiodination pathways that can metabolize T4 more than T3 appear to be involved, but specific activities need further investigation. Although a comparison of the different effects of each HBCD diasteroisomer are complicated by in vivo bioisomerization of the β- and γ-forms, thyroid axis effects appear to be most consistently associated with exposure to the γ-diasteroisomer.
Acknowledgments The authors acknowledge the Existing Substances Branch of Environment Canada for partially funding this project.
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